The discovery that Ras proteins are farnesylated and that inhibition of farnesylation prevents cellular transformation has generated an explosion of interest in this post-translational modification; currently, several drug candidates that target farnesyl transferase are in Phase 2 trials. However, despite this success, our current understanding of the enzymology of prenyltransferases remains incomplete particularly regarding the precise chemical mechanism. Moreover, many aspects concerning the biological function of protein prenylation remain unclear. In the first three years of this project, we have gained significant insights into the structure and mechanism of prenyltransferases; we have also made progress in understanding functional aspects of this modification. The specific aims of this renewal application are to: (1) Complete our studies of the chemical mechanisms of the reactions catalyzed by protein farnesyl and geranylgeranyl transferase enzymes. Stereochemical and kinetic isotope effect experiments will be performed. (2) Use the structural insights we have gained from our studies with isoprenoid analogues to design a new class of bisubstrate inhibitors of protein farnesyltransferase. (3) Identify additional proteins that recognize prenylated proteins and peptides through interactions with the isoprenoid. These will be identified using a photoaffinity capture method employing biotinylated prenylcysteines and prenylated peptides coupled with mass spectral analysis for target identification. These studies will determine how common this phenomenon is and evaluate how many different structural classes exist. (4) Use a combination of synthetic methods and phage display to obtain inhibitors that target the interaction between prenylated proteins and their receptors. Such inhibitors will be useful for clarifying the biological function of protein prenylation. Completion of the above aims should provide additional insights into the mechanism and function of protein prenylation.